Sequences and methods for detection of HIV-1

ABSTRACT

Primers and probes derived from the HIV-1 pol gene which facilitate detection and/or quantification of all presently known genotypes of HIV-1 (A-I and O). Disclosed sequences may be used in a variety of amplification and non-amplification formats for detection of HIV nucleic acids.

FIELD OF THE INVENTION

[0001] The present invention relates to materials and methods fordetection of HIV nucleic acids, in particular to probes and primers fordetection of HIV in hybridization and amplification assays.

BACKGROUND OF THE INVENTION

[0002] The genome of the Human Immunodeficiency Virus (HIV) is highlyheterogeneous and exhibits a mutation rate on the order of 10⁻⁴ per baseper generation. Combined with the rapid rate of viral propagation ininfected individuals, this presents particular challenges for diagnosticnucleic acid amplification techniques which typically amplify a singleconserved sequence within a target organism. Although a great deal ofresearch activity has been directed to detecting HIV-1 in hybridizationand amplification assays, such molecular assays have so far been limitedby their inability to detect all genotypes with equal efficiency.Although a signal amplification system for the detection of HIV-1 type Ohas been reported, none of the currently available diagnostic nucleicacid amplification methods are able to detect this genotype of thevirus.

SUMMARY OF THE INVENTION

[0003] The present invention provides primers and probes derived fromthe HIV-1 pol gene which facilitate detection and/or quantification ofall presently known genotypes of HIV-1 (A-I and O). A singleamplification primer pair according to the invention efficientlyamplifies all known genotypes of HIV-1, which may then be detected in asingle detection step using the detector probes and primers of theinvention.

DESCRIPTION OF THE DRAWINGS

[0004]FIG. 1 shows the titration of internal control fluorescent signal(FAM) with increasing levels of native HIV target (ROX).

DETAILED DESCRIPTION OF THE INVENTION

[0005] The primers, hybridization probes and detector probes of thepresent invention are complementary to regions of the HIV-1 polymerase(pol) gene. Initially, design of the disclosed primers and probes wasbased on conserved regions in an alignment of fifty-eight HIV-1 polsequences. Additional data was subsequently added to the alignment toprovide a database with a total of 115 pol sequences. One goal was todevelop probes and primers which, in spite of the heterogeneity of thepol sequence, provided amplification, detection and/or quantitation ofall presently known HIV-1 genotypes with essentially equal efficiency inamplification reactions. In some cases this was accomplished byoverlapping the hybridization site of the 5′ ends of certain of thedetector probes with the hybridization site of the 3′ end anamplification primer. This approach took advantage of sequenceconservation in the SDA primer region and avoided much of the sequenceheterogeneity evident in the intervening region between the two SDAprimers. This technique also allowed use of a smaller target sequence,thereby improving amplification efficiency.

[0006] As used herein, an amplification primer is an oligonucleotide foramplification of a target sequence by extension of the oligonucleotideafter hybridization to the target sequence or by ligation of multipleoligonucleotides which are adjacent when hybridized to the targetsequence. At least a portion of the amplification primer hybridizes tothe target. This portion is referred to as the target binding sequenceand it determines the target-specificity of the primer. In addition tothe target binding sequence, certain amplification methods requirespecialized non-target binding sequences in the amplification primer.These specialized sequences are necessary for the amplification reactionto proceed and typically serve to append the specialized sequence to thetarget. For example, the amplification primers used in SDA include arestriction endonuclease recognition site 5′ to the target bindingsequence (U.S. Pat. Nos. 5,270,184 and 5,455,166). NASBA, 3SR andtranscription based amplification primers require an RNA polymerasepromoter linked to the target binding sequence of the primer. Linkingsuch specialized sequences to a target binding sequence for use in aselected amplification reaction is routine in the art. In contrast,amplification methods such as PCR, which do not require specializedsequences at the ends of the target, generally employ amplificationprimers consisting of only target binding sequence.

[0007] As used herein, the terms “primer” and “probe” refer to thefunction of the oligonucleotide. A primer is typically extended bypolymerase or ligation following hybridization to the target but a probetypically is not. A hybridized oligonucleotide may function as a probeif it is used to capture or detect a target sequence, and the sameoligonucleotide may function as a primer when it is employed as a targetbinding sequence in an amplification primer. It will therefore beappreciated that any of the target binding sequences disclosed hereinfor amplification, detection or quantitation of HIV-1 may be used eitheras hybridization probes or as target binding sequences in primers fordetection or amplification, optionally linked to a specialized sequencerequired by the selected amplification reaction or to facilitatedetection.

[0008] Based on the alignment of multiple HIV-1 pol gene sequences, thefollowing amplification primers were designed for testing in SDAreactions. Target binding sequences are underlined. The remaining 5′portion of the sequence comprises the restriction endonucleaserecognition site (RERS) that is required for the SDA reaction to proceedand a generic non-target-specific tail sequence. It will be readilyapparent that the target binding sequences may be used alone to amplifythe target in reactions which do not require specialized sequences orstructures (e.g., PCR) and that different specialized sequences requiredby amplification reactions other than SDA may be substituted for theRERS-containing sequence shown below (e.g., an RNA polymerase promoter).“R” and “L” in the primer name indicates “right” and “left” primers,respectively, when the oligonucleotides are used in amplificationreactions: AMPLIFICATION PRIMERS QAL48CGATTCCGCTCCAGACTTCTCGGGTAGATACAGGAGCAGAT SEQ ID NO:1 QAL46CGATTCCGCTCCAGACTTCTCGGGAGATACAGGAGCAGAT SEQ ID NO:2 QAR48ACCGCATCGAATGCATGTCTCGGGCTATCATTTTTGGTTTCC SEQ ID NO:3 QAR44ACCGCATCGAATGCATGTCTCGGGTATCATTTTTGGTTTCC SEQ ID NO:4 AL46CGATTCCGCTCCAGACTTCTCGGGCAGTACAAATGGCAGT SEQ ID NO:5 AL48CGATTCCGCTCCAGACTTCTCGGGGCAGTACAAATGGCAG SEQ ID NO:6 AL50CGATTCCGCTCCAGACTTCTCGGGGCAGTACAAATGGCAGT SEQ ID NO:7 AR44ACCGCATCGAATGACTGTCTCGGGTGTACCCCCCAATC SEQ ID NO:8 AR44BACCGCATCGAATGACTGTCTCGGGCTGTACCCCCCAAT SEQ ID NO:9 AR48ACCGCATCGAATGACTGTCTCGGGTGTACCCCCCAATCC SEQ ID NO:10

[0009] In addition, the following detector primers were designed forreal-time detection of amplification products produced using theamplification primers. The structure and use of such detector primers isdescribed, for example, in U.S. Pat. No. 5,547,861 and U.S. Pat. No.5,928,869. The target binding sequences are underlined. The remainingportion of the sequence forms a hairpin structure which is typicallylabeled to facilitate detection of amplification products as is known inthe art. It will be readily apparent that the target sequence may beused alone for direct detection by hybridization (typically linked to adetectable label) and that other directly and indirectly detectablesequences and labels may be substituted for the hairpin as is known inthe art. See, for example U.S. Pat. No. 5,935,791; U.S. Pat. No.5,846,726; U.S. Pat. No. 5,691,145; U.S. Pat. No. 5,550,025 and U.S.Pat. No. 5,593,867. DETECTOR PRIMERS QDR66TAGCACCCGAGTGCTGGCAAATTCATTTCTTCTAATACTG SEQ ID NO:11 QDR64TAGCACCCGAGTGCTGCAAATTCATTTCTTCTAATACTGT SEQ ID NO:12 QDR56TAGCACCCGAGTGCTAAATTCATTTCTTCTAATACTGT SEQ ID NO:13 QOL1TAGCACCCGAGTGCTAGGAGCAGATGATACAGT SEQ ID NO:14 QOL2TAGCACCCGAGTGCTCAGGAGCAGATGATACAGT SEQ ID NO:15 QOL3TAGCACCCGAGTGCTACAGGAGCAGATGATACAGT SEQ ID NO:16 QOL4TAGCACCCGAGTGCTGAGCAGATGATACAGT SEQ ID NO:17 QOL5TAGCACCCGAGTGCTGGAGCAGATGATACAGT SEQ ID NO:18 DL56HPDTAGCACCCGAGTGCTCACAATGTTAAAAGAAAAGGG SEQ ID NO:19 DL52HPDTAGCACCCGAGTGCTACAATGTTAAAAGAAAAGGG SEQ ID NO:20 DL50HPDTAGCACCCGAGTGCTCAATGTTAAAAGAAAAGGG SEQ ID NO:21 DR58HPDTAGCACCCGAGTGCTCCCCTTTTCTATTAAAATTGTG SEQ ID NO:22 DR54HPDTAGCACCCGAGTGCTCCCTTTTCTATTAAAATTGTG SEQ ID NO:23 DR52HPDTAGCACCCGAGTGCTCCCCTTTTCTATTAAAATTG SEQ ID NO:24 DR48HPDTAGCACCCGAGTGCTCCCTTTTCTATTAAAATTG SEQ ID NO:25 Pol2DR58TAGCACCCGAGTGCTCCCCTTTTCTTTTAAAATTGTG SEQ ID NO:26 DN2TAGCACCCGAGTGCTCCCAATCCCCCCTTTTCTGTTAAAAT SEQ ID NO:27 DN3TAGCACCCGAGTGCTCCCCAATCCCCCCTTTTCTGTTAAAAT SEQ ID NO:28 DN4TAGCACCCGAGTGCTCCAATCCCCCCTTTTCTGTTAAAAT SEQ ID NO:29 DN5TAGCACCCGAGTGCTCAATCCCCCCTTTTCTGTTAAAAT SEQ ID NO:30 DN5.1TAGCACCCGAGTGCTCAATCCCCCCTTTTCTTTTAAAAT SEQ ID NO:31 OL62TAGCACCCGAGTGCTCCCAATCCCCCCTTTTCTTTT SEQ ID NO:32 OL64TAGCACCCGAGTGCTCCCAATCCCCCCTTTTCTTTTA SEQ ID NO:33

[0010] SEQ ID NOs:11-13 and 19-26 are conventional non-overlappingdetector primers which contain a hairpin as described in U.S. Pat. No.5,928,869. SEQ ID NOs:14-18 and 27-33 also contain the hairpin but the5′ end of the target binding sequences overlap with the 3′ end of thetarget binding sequences of the upstream amplification primers.

[0011] Bumper primers used in SDA (BR and BL) were also designed. Theentire sequence of these oligonucleotides consists of target bindingsequence: BUMPER PRIMERS/DETECTOR PROBES QBL44 CTAAAGGAAGCTCTAT SEQ IDNO:34 QBR42 AACCTCCAATTCCC SEQ ID NO:35 BL54 GAATCTATGAATAAAGAATTAAA SEQID NO:36 BR54 TGCTATTATGTCTACTATTCT SEQ ID NO:37

[0012] The sequences set forth above were selected to minimize theeffects of heterogeneity in the targeted region of the pol gene.Mismatches were confined to the middle or the 5′ end of the sequences topermit efficient 3′ extension upon hybridization to the target sequence.Only three of the 117 HIV-1 strains analyzed exhibit more than onemismatch with amplification primers SEQ ID NO:5 and SEQ ID NO:9 or withdetector SEQ ID NO:26. Detector SEQ ID NO:27-30 contain a deliberatemismatch eight bases from the 3′ end to minimize target-specificvariations in priming efficiency caused by heterogeneity in the targetregion.

[0013] Because the target binding sequence confers target specificity onthe primer or probe, it should be understood that the target bindingsequences exemplified above for use as particular components of aspecific amplification reaction may also be used in a variety of otherways for detection of HIV. For example, the target binding sequences ofSEQ ID NOs:1-37 may alternatively be used as hybridization probes fordirect detection of HIV-1, either without prior amplification or as apost-amplification assay. Such hybridization methods are well known inthe art and typically employ a detectable label associated with orlinked to the target binding sequence to facilitate detection ofhybridization. Further, essentially all of the target binding sequencesset forth above may be used as amplification primers in amplificationreactions which do not require additional specialized sequences (such asPCR) or appended to the appropriate specialized sequences for use in3SR, NASBA, transcription-based or any other primer extensionamplification reactions. For detection of amplification products,amplification primers comprising the target binding sequences disclosedherein may be labeled as is known in the art, or labeled detectorprimers comprising the disclosed target binding sequences may be used inconjunction with the amplification primers as described in U.S. Pat. No.5,547,861 and U.S. Pat. No. 5,928,869 for real-time homogeneousdetection of amplification. Such detector primers typically comprise adirectly or indirectly detectable sequence which does not initiallyhybridize to the target but which facilitates detection of the detectorprimer once it has hybridized to the target and been extended. Forexample, such detectable sequences may be sequences which form asecondary structure, sequences which contain a restriction site, orlinear sequences which are detected by hybridization of theircomplements to a labeled oligonucleotide (sometimes referred to as areporter probe) as is known in the art. Alternatively, the amplificationproducts may be detected post-amplification by hybridization of a probeselected from any of the target binding sequences disclosed herein whichfall between a selected set of amplification primers.

[0014] It is to be understood that an oligonucleotide according to theinvention which consists of a target binding sequence and, optionally,either a sequence required for a selected amplification reaction or asequence required for a selected detection reaction may also includecertain other sequences which serve as spacers, linkers, sequences forlabeling or binding of an enzyme, etc. Such additional sequences aretypically known to be necessary to obtain optimum function of theoligonucleotide in the selected reaction and are intended to be includedby the term “consisting of.”

EXAMPLE 1

[0015] SDA reactions were performed to determine the analyticalsensitivity of the assay for the detection of HIV DNA. Amplification wascarried in the presence of 0, 5, 10, 50, 100 or 250 copies of HIV targetDNA. The target sequence was a fragment of the HIV-1 genomecorresponding to nucleotides 4659-4910 of strain B-WEAU (GenBankaccession number U21135) that was cloned into the plasmid vectorpBlueScript SK+ (Stratagene). SDA was performed at 52° C. using 500 nMprimers (SEQ ID NO:5 and SEQ ID NO:9), 50 nM bumpers (SEQ ID NO:36 andSEQ ID NO:37), and 200 nM detector probe, SEQ ID NO:26, in buffercontaining: 100 mM bicine; 30 mM potassium hydroxide; 68 mM K_(i)PO₄,pH7.6; 10.5% glycerol; 6.5% DMSO; 0.7 mM dCTP; 0.1 mM dA-, dG- and dTTP;70 ng/μl human placental DNA; 100 ng/μl bovine serum albumin; 4 mMmagnesium acetate; 6U Bst polymerase and 32U BsoBI restriction enzyme.The detector primer was labeled at the 5′ terminus with a fluoresceindonor molecule and internally with a dabcyl quencher moiety. The twodyes on the detector were separated by BsoBI restriction endonucleaserecognition sequence and held in close juxtaposition by a hairpinstructure within the tail sequence as described in U.S. Pat. Nos.5,919,630; 5,928,869 and 5,958,700. Donor fluorescence was monitoredduring the course of amplification.

[0016] In the presence of target, donor fluorescence increased duringthe course of the reaction as the hairpin holding the donor and quencherin close proximity unfolded and the restriction site was cleaved. Incontrast, in the absence of target, fluorescence remained consistentlylow throughout the reaction. Results were expressed in terms of areaunder the curve or “MOTA.” A larger MOTA score indicates generation ofmore fluorescence and, generally, the presence of more input target. Theresults are shown in Table 1. TABLE 1 TARGETS PER MEAN MOTA REACTIONSCORE (n = 28)*  0  606  5 14883 10 20565 50 47643 100  56740 150  61676

[0017] The limit of detection (LOD), defined as the input target levelat which 95% of reactions would yield a positive result, was determinedto be between 9 and 11 copies of target DNA depending on the MOTA scoreselected as a cut-off for determining a positive result. These resultsdemonstrate that the amplification and detector primers are capable ofsensitive and reproducible detection of HIV DNA.

EXAMPLE 2

[0018] A similar experiment to that described in Example 1 was performedusing the SEQ ID NO:27 detector primer, the target hybridization regionof which overlaps the SEQ ID NO:9 amplification primer. This probe wasdesigned to take advantage of sequence conservation in the amplificationprimer binding region while maintaining the specificity afforded bydetection of the internal region of the SDA amplicon that lies betweenthe two amplification primers. Buffer conditions were the same asdescribed above with the following modifications: 75 mM KPO4, 14% DMSO,5% glycerol and 27U BsoBI. The limit of detection using the overlappingprobe was 61 to 86 copies of HIV target DNA depending on the MOTA scoreselected for determining a positive result. These results demonstratesensitive and specific detection of HIV target DNA using an alternativeprobe design that offers additional flexibility in the development ofSDA-based systems.

EXAMPLE 3

[0019] The SDA assay for HIV DNA was converted to a two-step reversetranscriptase (RT)-SDA format in which RNA was first copied to cDNAusing an RT enzyme and then amplified in a conventional SDA reaction.One important difference between the SDA conditions described inExamples 1 and 2 and the RT-SDA described here is the absence of bumperprimers. The need for bumpers apparently was precluded by the choice ofan RT enzyme which possesses RNase H activity and which is thereforecapable of degrading the RNA template following reverse transcription,thereby liberating a single stranded DNA target sequence in to solution.An LOD study was performed for HIV RNA using in vitro transcripts of agenotype B strain generated from the pBlueScript plasmid clone describedin Example 1.

[0020] Reverse transcription was carried out in microtiter wells using15U avian myeloblastosis virus (AMV)-RT in buffer containing: 69.3 mMbicine; 12.3 mM KOH; 20.8 mM K_(i)PO₄; 3% glycerol; 4.5% DMSO; 6 mMmagnesium acetate; 2.75 mM dCsTP; 0.25 mM dA-, dG- and DTTP; 100 ng/μlBSA; 1250 nM SEQ ID NO:5 and 700 nM SEQ ID NO:9. In brief, the reversetranscription reaction mixture without enzyme was incubated for 10 minat 68° C. to denature the target RNA. Eighty microliters of denaturedtarget was then added to a microwell containing 20 μl of an RT enzymemixture that was pre-equilibrated at 50° C. The complete RT reaction wasincubated for 10 min to facilitate synthesis of cDNA and degradation ofthe RNA template by the RNase H activity of the RT enzyme. To initiateamplification, 40 μl of the reverse transcription reaction wastransferred to a second microtiter well at 52° C. containing 60 μl of anamplification mixture comprising Bstpolymerase, BsoBI restrictionenzyme, and fluorescent detector primer together with certain buffercomponents. The final SDA conditions were as follows: 72 mM bicine; 24mM potassium hydroxide; 54.4 mM K_(i)PO₄; 4% glycerol; 11% DMSO; 0.1 mMdA-, dG- and dTTP; 1.1 mM dCTP; 700 ng/μl hpDNA; loong/μl BSA; 500 nMSEQ ID NO:5; 300 nM SEQ ID NO:9; 600 nM SEQ ID NO:26; 12U BsoBI and 7UBst polymerase. The wells were sealed and incubated at 52° C. Donorfluorescence was monitored throughout the course of the reaction.

[0021] The results of this experiment are shown in Table 2. TABLE 2 RNATARGETS MEAN MOTA PER REACTION SCORE (n = 17) % POSITIVE  0  139  0   10 3577  64.7 25  7810  88.0 50 13410 100.0 75 23641 100.0 100  35719100.0 500  73870 100.0

[0022] MOTA scores ≧1000 were considered positive . All reactionscontaining ≧50 copies of HIV target RNA were positive as were 88%containing 25 copies and 65% of those containing 10 copies. Theseresults demonstrate the sensitivity of the disclosed amplification anddetector primers for the detection of HIV-1 RNA.

EXAMPLE 4

[0023] RT-SDA was performed on purified RNA from representative isolatesof nine different clades of HIV-1 (Boston Biomedica, Inc.). In order toquantify the parental stocks of viral RNA, all except the Type O nucleicacid were tested with each of two commercially available quantitativeHIV tests: the Roche Amplicor HIV-1 Monitor V1.5 and the ChironQuantiplex HIV-1 RNA 3.0 Assay. Since neither of these tests is able toamplify RNA of HIV-1 genotype O, a third non-amplified system, theDigene Hybrid Capture Assay, was used to quantify the type O nucleicacid. Viral RNAs for genotypes A-H were diluted according to the resultsof the Roche assay to give the equivalent of 200 copies per RT-SDAreaction. Type O RNA was diluted to the same level based on the resultsof the Digene test. RT-SDA was performed according to the methoddescribed in Example 3. For eight of the nine clades, all sixteenreplicates were positive at 200 copies per RT-SDA reaction. For genotypeF, {fraction (4/16)} (25%) replicates were positive. The reason for theapparently lower sensitivity with type F is unclear but might beattributed to inaccurate qualification of the parental RNA stock of thisgenotype by the Roche assay. Alternatively, the discrepancy might be dueto the presence in the type F sequence of a single base mismatch withthe right amplification primer which is used to initiate first strandcDNA synthesis (SEQ ID NO:9). Importantly, all reactions conducted with2000 input copies of type F RNA were psotive.

[0024] These data demonstrate that the disclosed primers and probes arecapable of detecting multiple genotypes of HIV-1, including type O, witha high degree of sensitivity. Detection of type O RNA is particularlyimportant in view of the inability to detect this lade with the majorityof commercially available diagnostic nucleic acid assays.

EXAMPLE 5

[0025] In vitro transcripts generated from the plasmid clone describedin Example 1 were quantified by competitive RT-SDA. In brief, RT-SDA wasperformed as described above with the exception that two detectorprimers were included in the reaction mixture, both at a concentrationof 200 nM. The first primer, SEQ ID NO:26, was specific for HIV-1 andwas labeled at the 5′ end with dabcyl quencher moiety and internallywith rhodamine (Rox). The second probe was specific for an internalcontrol sequence and was labeled at its 5′ end with fluorescein andinternally with a dabcyl quencher. The internal control comprised an RNAmolecule that was generated by in vitro transcription of a mutated cloneof the HIV-1 pol gene. The internal control possessed the same primerbinding regions as the native HIV target but differed by a series ofpoint mutations introduced to coincide with the 3′ end of the detectorhybridization region. These mutations permitted discrimination of thenative target and internal control by preventing hybridization andextension of the mismatched probes during amplification. The internalcontrol was designed such that it amplified with similar efficiency tothe native target and would compete during the course of the reactionfor one or more rate-limiting reagents. The same amount of internalcontrol was seeded into each test sample and into a series of calibratorreactions containing known amounts of native target. Details of thetheoretical aspects of the quantitative SDA algorithm may be foundelsewhere (J. G. Nadeau, et al. 1999. Anal Biochem 276, 177-187; C. M.Nycz, et al. 1998. Anal Biochem 259, 226-234). In brief, thefluorescence produced by amplification of both the native target andinternal control was monitored at discreet intervals throughout thereaction. Data were processed through a series of normalization andsmoothing functions to produce a fluorescent signal for both sequences.From these values, the natural log of the ratio of native target andinternal control signals was calculated. The values obtained from thecalibrator wells were plotted as a regression against the natural log ofthe input number of target molecules of RNA to produce calibrationcurves corresponding to each time point. An automated algorithm was thenused to determine the time at which it was statistically optimal toperform quantification.

[0026]FIG. 1 shows the MOTA scores for the internal control with variouscopy numbers of input target sequence. MOTA scores for the internalcontrol decreased with increasing levels of native target, demonstratingthe competition between the two targets that is necessary for accuratequantification. Table 3 shows the results of quantitative competitiveRT-SDA with between 50 and 100000 copies of native RNA target and 5000copies of internal control per reaction. At all but one target level,accuracy and precision were better than ±25%, thus demonstrating theability to quantify HIV-1 RNA using the disclosed amplification anddetector primer sequences. TABLE 3 TARGET RNA COPIES/REACTION REVERSEPERCENT TRANSCR. SDA* MEAN (n = 8) ACCURACY PRECISION   0    0   1662.78  125   50   47 −5.17 25.47  1250   500  537 7.45 24.19 12500  5000 8765 75.29 9.07 125000   50000 60009 20.02 5.48 250000  100000 76351−23.65 7.26

[0027] Sequence alignment data and initial testing of the additionaltarget binding sequences disclosed herein indicates that similar resultswould be obtained using these sequences in probes and primers foramplification and/or detection of all genotypes of HIV-1.

1 37 1 41 DNA Human immunodeficiency virus type 1 1 cgattccgctccagacttct cgggtagata caggagcaga t 41 2 40 DNA Human immunodeficiencyvirus type 1 2 cgattccgct ccagacttct cgggagatac aggagcagat 40 3 42 DNAHuman immunodeficiency virus type 1 3 accgcatcga atgcatgtct cgggctatcatttttggttt cc 42 4 41 DNA Human immunodeficiency virus type 1 4accgcatcga atgcatgtct cgggtatcat ttttggtttc c 41 5 40 DNA Humanimmunodeficiency virus type 1 5 cgattccgct ccagacttct cgggcagtacaaatggcagt 40 6 40 DNA Human immunodeficiency virus type 1 6 cgattccgctccagacttct cggggcagta caaatggcag 40 7 41 DNA Human immunodeficiencyvirus type 1 7 cgattccgct ccagacttct cggggcagta caaatggcag t 41 8 38 DNAHuman immunodeficiency virus type 1 8 accgcatcga atgactgtct cgggtgtaccccccaatc 38 9 38 DNA Human immunodeficiency virus type 1 9 accgcatcgaatgactgtct cgggctgtac cccccaat 38 10 39 DNA Human immunodeficiency virustype 1 10 accgcatcga atgactgtct cgggtgtacc ccccaatcc 39 11 40 DNA Humanimmunodeficiency virus type 1 11 tagcacccga gtgctggcaa attcatttcttctaatactg 40 12 40 DNA Human immunodeficiency virus type 1 12tagcacccga gtgctgcaaa ttcatttctt ctaatactgt 40 13 38 DNA Humanimmunodeficiency virus type 1 13 tagcacccga gtgctaaatt catttcttctaatactgt 38 14 33 DNA Human immunodeficiency virus type 1 14 tagcacccgagtgctaggag cagatgatac agt 33 15 34 DNA Human immunodeficiency virus type1 15 tagcacccga gtgctcagga gcagatgata cagt 34 16 35 DNA Humanimmunodeficiency virus type 1 16 tagcacccga gtgctacagg agcagatgat acagt35 17 31 DNA Human immunodeficiency virus type 1 17 tagcacccgagtgctgagca gatgatacag t 31 18 32 DNA Human immunodeficiency virus type 118 tagcacccga gtgctggagc agatgataca gt 32 19 36 DNA Humanimmunodeficiency virus type 1 19 tagcacccga gtgctcacaa tgttaaaaga aaaggg36 20 35 DNA Human immunodeficiency virus type 1 20 tagcacccgagtgctacaat gttaaaagaa aaggg 35 21 34 DNA Human immunodeficiency virustype 1 21 tagcacccga gtgctcaatg ttaaaagaaa aggg 34 22 37 DNA Humanimmunodeficiency virus type 1 22 tagcacccga gtgctcccct tttctattaaaattgtg 37 23 36 DNA Human immunodeficiency virus type 1 23 tagcacccgagtgctccctt ttctattaaa attgtg 36 24 35 DNA Human immunodeficiency virustype 1 24 tagcacccga gtgctcccct tttctattaa aattg 35 25 34 DNA Humanimmunodeficiency virus type 1 25 tagcacccga gtgctccctt ttctattaaa attg34 26 37 DNA Human immunodeficiency virus type 1 26 tagcacccgagtgctcccct tttcttttaa aattgtg 37 27 41 DNA Human immunodeficiency virustype 1 27 tagcacccga gtgctcccaa tccccccttt tctgttaaaa t 41 28 42 DNAHuman immunodeficiency virus type 1 28 tagcacccga gtgctcccca atccccccttttctgttaaa at 42 29 40 DNA Human immunodeficiency virus type 1 29tagcacccga gtgctccaat cccccctttt ctgttaaaat 40 30 39 DNA Humanimmunodeficiency virus type 1 30 tagcacccga gtgctcaatc cccccttttctgttaaaat 39 31 39 DNA Human immunodeficiency virus type 1 31 tagcacccgagtgctcaatc cccccttttc ttttaaaat 39 32 36 DNA Human immunodeficiencyvirus type 1 32 tagcacccga gtgctcccaa tccccccttt tctttt 36 33 37 DNAHuman immunodeficiency virus type 1 33 tagcacccga gtgctcccaa tccccccttttctttta 37 34 16 DNA Human immunodeficiency virus type 1 34 ctaaaggaagctctat 16 35 14 DNA Human immunodeficiency virus type 1 35 aacctccaattccc 14 36 23 DNA Human immunodeficiency virus type 1 36 gaatctatgaataaagaatt aaa 23 37 21 DNA Human immunodeficiency virus type 1 37tgctattatg tctactattc t 21

What is claimed is:
 1. A method for detecting an HIV-1 target sequencecomprising: a) amplifying the target sequence using a firstamplification primer having a sequence consisting of the target bindingsequence of any one of SEQ ID NO: 1 through SEQ ID NO:37 and,optionally, a sequence required for a selected amplification reaction,and; b) detecting the amplified target sequence.
 2. The method of claim1 further comprising a second amplification primer having a sequenceconsisting of the target binding sequence of any one of SEQ ID NO: 1through SEQ ID NO:37 and, optionally a sequence required for a selectedamplification reaction.
 3. The method of claim 1 wherein the targetbinding sequence of the first amplification primer is the target bindingsequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:6 or SEQ IDNO:7.
 4. The method of claim 3 wherein the first amplification primer isselected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:5, SEQ ID NO:6 and SEQ ID NO:7.
 5. The method of claim 2 wherein thetarget binding sequence of the second amplification primer is the targetbinding sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:9or SEQ ID NO:10.
 6. The method of claim 5 wherein the secondamplification primer is selected from the group consisting of SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10.
 7. Themethod of claim 1 wherein the amplified target sequence is detectedusing an oligonucleotide having a sequence consisting of the targetbinding sequence of any one of SEQ ID NO:11 through SEQ ID NO:33 and,optionally, a sequence required for a selected detection reaction. 8.The method of claim 7 wherein the oligonucleotide is selected such thata 5′ end of the target binding sequence of the oligonucleotide fordetection overlaps a 3′ end of the target binding sequence of the firstamplification primer.
 9. The method of claim 7 wherein the targetbinding sequence of the oligonucleotide is the target binding sequenceof any one of SEQ ID NO:11 through SEQ ID NO:33.
 10. The method of claim9 wherein the oligonucleotide is selected from the group consisting ofany one of SEQ ID NO:11 through SEQ ID NO:33.
 11. The method of claim 7wherein the sequence required for the selected detection reaction is ahairpin, G-quartet, restriction site or a sequence which hybridizes to areporter probe.
 12. The method of claim 7 wherein the oligonucleotidecomprises a detectable label.
 13. The method of claim 12 wherein thelabel is a fluorescent label.
 14. The method of claim 7 wherein theoligonucleotide is unlabeled and the target sequence is detected byhybridization of the oligonucleotide to a labeled reporter probe. 15.The method of claim 1 further comprising quantifying the targetsequence.
 16. The method of claim 15 wherein the target sequence isquantified by coamplification of a control sequence and the targetsequence.
 17. The method of claim 16 wherein the coamplification of thetarget and control sequences is competitive and detected in real-time.18. The method of claim 16 wherein coamplification is detected in ahomogeneous assay.
 19. The method of claim 1 wherein multiple HIV-1genotypes are detected.
 20. An oligonucleotide having a sequenceconsisting of the target binding sequence of any one of SEQ ID NO:1through SEQ ID NO:37 and, optionally, either a sequence required for aselected amplification reaction or a sequence required for a selecteddetection reaction.
 21. The oligonucleotide of claim 20 which consistsof the target binding sequence of any one of SEQ ID NO:1 through SEQ IDNO:10 and, optionally, a sequence required for a selected amplificationreaction.
 22. The oligonucleotide of claim 21 selected from the groupconsisting of any one of SEQ ID NO:1 through SEQ ID NO:10.
 23. Theoligonucleotide of claim 20 which consists of the target bindingsequence of any one of SEQ ID NO:11 through SEQ ID NO:33 and,optionally, a sequence required for a selected detection reaction. 24.The oligonucleotide of claim 23 selected from the group consisting ofany one of SEQ ID NO:11 through SEQ ID NO:33.
 25. The oligonucleotide ofclaim 23 wherein the sequence required for the detection reaction is ahairpin, a G-quartet, a restriction site or a sequence which hybridizesto a reporter probe.
 26. The oligonucleotide of claim 23 which islabeled with a detectable label.
 27. The oligonucleotide of claim 26wherein the label is a fluorescent label.